P-selectin (CD62, GMP-140, PADGEM), a Ca2+-dependent lectin on activated platelets and endothelium, functions as a receptor for myeloid cells by interacting with sialylated, fucosylated lactosaminoglycans. P-selectin binds to a limited number of protease-sensitive sites on myeloid cells, but the protein(s) that carry the glycans recognized by P-selectin are unknown. Blotting of neutrophil or HL-60 cell membrane extracts with [125I] P-selectin and affinity chromatography of [3H] glucosamine-labeled HL-60 cell extracts were used to identify P-selectin ligands. A major ligand was identified with an approximately 250,000 Mr under nonreducing conditions and approximately 120,000 under reducing conditions. Binding of P-selectin to the ligand was Ca2+ dependent and was blocked by mAbs to P-selectin. Brief sialidase digestion of the ligand increased its apparent molecular weight; however, prolonged digestion abolished binding of P-selectin. Peptide:N-glycosidase F treatment reduced the apparent molecular weight of the ligand by approximately 3,000 but did not affect P-selectin binding. Western blot and immunodepletion experiments indicated that the ligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Lex, nor was it leukosialin, a heavy sialylated glycoprotein of similar molecular weight. The preferential interaction of the ligand with P-selectin suggests that it may play a role in adhesion of myeloid cells to activated platelets and endothelial cells.
The selectins are three structurally related membrane glycoproteins that participate in leukocyte adhesion to vascular endothelium and platelets, as reviewed by McEver, xe2x80x9cLeukocyte interactions mediated by selectingxe2x80x9d Thromb. Haemostas. 66:80-87 (1991). P-selectin (CD62), previously known as GMP-140 or PAD-GEM protein, is a receptor for neutrophils, monocytes and subsets of lymphocytes that is rapidly translocated from secretory granule membranes to the plasma membrane of activated platelets, as reported by Hamburger and McEver, xe2x80x9cGMP 140 mediates adhesion of stimulated platelets to neutrophilsxe2x80x9d Blood 75:550-554 (1990); Larsen et al., xe2x80x9cPADGEM protein: a receptor that mediates the interaction of activated platelets with neutrophils and monocytesxe2x80x9d Cell 59:305-312 (1989) and endothelial cells, as reported by Geng et al., xe2x80x9cRapid neutrophil adhesion to activated endothelium mediated by GMP-140xe2x80x9d Nature 343:757-760 (1990); Lorant et al., xe2x80x9cCoexpression of GMP 140 and PAF by endothelium stimulated by histamine or thrombin: A juxtacrine system for adhesion and activation of neutrophilsxe2x80x9d J. Cell Biol. 115:223-234 (1991).
E-selectin (ELAM-1) is a cytokine-inducible endothelial cell receptor for neutrophils, as reported by Bevilacqua et al., xe2x80x9cIdentification of an inducible endothelial leukocyte adhesion moleculexe2x80x9d Proc. Natl. Acad. Sci. USA 84:9238-9242 (1987), monocytes, as reported by Hession et al., xe2x80x9cEndothelial leukocyte adhesion molecule 1: Direct expression cloning and functional interactionsxe2x80x9d Proc. Natl. Acad. Sci. USA 87:1673-1677 (1990), and memory T cells, as reported by Picker et al., xe2x80x9cELAM-1 is an adhesion molecule for skin homing T cellsxe2x80x9d Nature (London) 349:796-799 (1991); Shimizu et al., xe2x80x9cActivation-independent binding of human memory T cells to adhesion molecule ELAM 1xe2x80x9d Nature (London) 349:799-802 (1991). L-selectin (LAM-1, LECAM-1), a protein expressed on myeloid cells and most lymphocytes, participates in neutrophil extravasation into inflammatory sites and homing of lymphocytes to peripheral lymph nodes, as reported by Lasky et al., xe2x80x9cCloning of a lymphocyte homing receptor reveals a lectin domainxe2x80x9d Cell 56:1045-1055 (1989); Siegelman et al., xe2x80x9cMouse lymph node homing receptor cDNA clone encodes a glycoprotein revealing tandem interaction domainsxe2x80x9d Science (Wash. DC) 243:1165-1172 (1989); Kishimoto et al., xe2x80x9cNeutrophil Mac-1 and MEL-1 adhesion proteins inversely regulated by chemotactic factorsxe2x80x9d Science (Wash. DC) 245:1238-1241 (1989); Watson et al., xe2x80x9cNeutrophil influx into an inflammatory site inhibited by a soluble homing receptor IgG chimaeraxe2x80x9d Nature (London) 349:164-167 (1991).
Each selectin functions as a CA2+-dependent lectin by recognition of sialylated glycans. Both E- and P-selectin interact with sialylated, fucosylated lactosaminoglycans on opposing cells, including the sialyl Lex tetrasaccharide, as reported by Phillips et al., xe2x80x9cELAM 1 mediates cell adhesion by recognition of a carbohydrate ligand, sialy-Lexxe2x80x9d Science (Wash DC) 250:1130-1132 (1990); Walz et al., xe2x80x9cRecognition by ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cellsxe2x80x9d Science (Wash. DC) 250:1132-1135 (1990); Lowe et al., xe2x80x9cELAM 1 dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNAxe2x80x9d Cell 63:475-484 (1990); Tiemeyer et al., xe2x80x9cCarbohydrate ligands for endothelial-leukocyte adhesion molecule 1xe2x80x9d Proc. Natl. Acad. Sci. USA 88:1138-1142 (1991); Goelz et al., xe2x80x9cELFT: a gene that directs the expression of an ELAM-1 ligandxe2x80x9d Cell 63:1349-1356 (1990); Polley et al., xe2x80x9cCD62 and endothelial cell-leukocyte adhesion molecule 1 (ELAM 1) recognize the same carbohydrate ligand sialyl-Lewis xxe2x80x9d Proc. Natl. Acad. Sci. USA 88:6224-6228 (1991); Zhou et al., xe2x80x9cThe selectin GMP-140 binds to sialyated, fucosylated lactosaminoglycans on both myeloid and nonmyeloid cellsxe2x80x9d J. Cell Biol. 115:557-564 (1991). However, the precise carbohydrate structures on myeloid cells recognized by these two selectins under physiologic conditions are not known. Such ligands might have unique structural features that enhance the binding specificity and/or affinity for their respective receptors.
P-selectin isolated from human platelets binds with apparent high affinity to a limited number of sites on neutrophils (Moore et al., xe2x80x9cGMP 140 binds to a glycoprotein receptor on human neutrophils: evidence for a lectin-like interactionxe2x80x9d J. Cell Biol. 112:491-499 (1991); Skinner et al., xe2x80x9cGMP-140 binding to neutrophils is inhibited by sulfated glycansxe2x80x9d. J. Biol. Chem. 266:5371-5374 (1991) and HL-60 cells (Zhou et al., xe2x80x9cThe selectin GMP-140 binds to sialyated, fucosylated lactosaminoglycans on both myeloid and nonmyeloid cellsxe2x80x9d J. Cell Biol. 115:557-564 (1991)). Binding is abolished by treatment of the cells with proteases (Moore et al., 1991), suggesting that the glycans on myeloid cells recognized preferentially by P-selectin are on glycoprotein(s) rather than on glycolipids. The number of binding sites for platelet P-selectin on neutrophils has been estimated at 10,000-20,000 per cell (Moore et al., 1991; Skinner et al., 1991), suggesting that these sites constitute a small component of the total cell surface protein. The protein portion of this ligand(s) may be crucial for binding by presenting the glycan in an optimal configuration, clustering glycans to enhance avidity, favoring the formation of specific oligosaccharide structures by cellular glycosyltransferases or modifying enzymes, and/or stabilizing the lectin-carbohydrate interaction through protein-protein interactions with P-selectin.
The potential importance of protein components in enhancing ligand affinity is supported by studies of. CHO cells transfected with a specific fucosyltransferase (Zhou et al., 1991). These cells express higher amounts of the sialyl Lex antigen than do HL-60 cells and have protease-sensitive binding sites for P-selectin. However, the interaction of P-selectin with these sites is of much lower apparent affinity than with those on myeloid cells, and adhesion of transfected CHO cells to immobilized P-selectin is weaker than that of neutrophils and HL-60 cells (Zhou et al., 1991). These observations suggest that myeloid cells express one or more membrane glycoproteins not found on CHO cells that enhance the lectin-mediated interaction with P-selectin. Alternatively, myeloid cells may express a glycosyltransferase or modifying enzyme not present in CHO cells.
It is therefore an object of the present invention to identify and characterize a specific glycoprotein ligand for P-selectin (CD62).
It is a further object of the present invention to provide methods and compositions derived from the characterization of a specific glycoprotein ligand for P-selectin for use in modifying inflammatory processes and in diagnostic assays.
P-selectin has been demonstrated to bind primarily to a single major glycoprotein ligand on neutrophils and HL-60 cells, when assessed by blotting assays and by affinity chromatography of [3H] glucosamine-labeled HL-60 cell extracts on immobilized P-selectin. This molecule was characterized and distinguished from other well-characterized neutrophil membrane proteins with similar apparent molecular mass.
The purified ligand, or fragments thereof (including both the carbohydrate and protein components), or antibodies to the ligand, or fragments thereof, can be used as inhibitors of binding of P-selectin to cells.
U.S. Ser. No. 07/554,199 filed Jul. 17, 1990 entitled xe2x80x9cPeptides Selectively Interacting with Selectinsxe2x80x9d by Rodger P. McEver, described the ability of P-selectin (GMP-140) to mediate cell-cell contact by binding to carbohydrate ligands on target cells and specific binding to protease-sensitive sites on human neutrophils. Studies with antibodies and with neuraminidase indicated that P-selectin bound to carbohydrate structures related to sialylated, fucosylated lactosaminoglycans. As described in U.S. Ser. No. 07/650,484 entitled xe2x80x9cLigand for GMP-140 Selectin and Methods of Use Thereofxe2x80x9d filed Feb. 5, 1991 by Rodger P. McEver, P-selectin was also demonstrated to bind to sialylated, fucosylated lactosaminoglycans (including the tetrasaccharide sialyl Lewis x (sLex)) on both myeloid and nonmyeloid cells.
The ability of proteases to abolish P-selectin binding to neutrophils indicated that high affinity binding of P-selectin to myeloid cells occurred through interactions with cell surface glycoprotein(s) rather than with glycolipids. As also described in U.S. Ser. No. 07/650,484, P-selectin bound preferentially to a glycoprotein in human neutrophil extracts of Mr 120,000, as analyzed by SDS-PAGE under reducing conditions. The glycoprotein was partially purified on a P-selectin affinity column. It appeared to be heavily glycosylated because it stained poorly with silver and Coomassie blue. It appeared to be heavily sialylated because it bound to a wheat germ agglutinin affinity column. Treatment of the glycoprotein ligand with low doses of sialidase slowed its mobility on SDS gels, a pattern consistent with partial desialylation of heavily O-glycosylated proteins. Binding of P-selectin to the glycoprotein ligand was Ca2+-dependent, blocked by monoclonal antibodies to P-selectin that also block P-selectin binding to leukocytes, and abolished by extensive treatment of the ligand with sialidase.
The preferential binding of P-selectin to the 120,000 D glycoprotein ligand in myeloid cell extracts suggested that it contained special structural features that are recognized with high affinity by P-selectin. Such structures might not be present on every protein or lipid characterized by sialylated, fucosylated structures such as sLex. It has now been further demonstrated that the adhesion of myeloid cells to immobilized P-selectin is much stronger than that to NeoLewis CHO cells (a cell line expressing sialylated, fucosylated lactosaminoglycans, described in U.S. Ser. No. 07/650,484), even though the NeoLewis cells express higher levels of sLex antigen, as reported by Zhou et al., J. Cell Biol. 115:557-564 (1991). Furthermore, fluid-phase [125I ]P-selectin binds with high affinity to a limited number of sites on myeloid cells, whereas it binds with lower affinity to a higher number of sites on NeoLewis CHO cells. The 120,000 D glycoprotein ligand for P-selectin in neutrophil extracts is likely to correspond to the limited number of protease-sensitive, high affinity binding sites for P-selectin on intact neutrophils. Interaction of P-selectin with these sites may be required for efficient adhesion of leukocytes in flowing blood to P-selectin expressed by activated platelets or endothelial cells.
Further structural features of the glycoprotein ligand for P-selectin and a method for purifying the ligand are described below. The purified ligand, or fragments thereof (including both the carbohydrate and protein components), or antibodies to the ligand, or fragments thereof, can be used as inhibitors of binding of P-selectin to cells.